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Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii.

Hwang S, Cordova B, Chavarria N, Elbanna D, McHugh S, Rojas J, Pfeiffer F, Maupin-Furlow JA - BMC Microbiol. (2014)

Bottom Line: The THI4 homolog of the halophilic archaea, including Hfx. volcanii (HVO_0665, HvThi4) was found to differ from that of methanogens and thermococci by having a cysteine residue (Cys165) corresponding to the conserved active site cysteine of yeast THI4p (Cys205).Based on our results, we conclude that the archaeon Hfx. volcanii uses a yeast THI4-type mechanism for sulfur relay to form the thiazole ring of thiamine.Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, 32611-0700, USA. sungminhwang@ufl.edu.

ABSTRACT

Background: Thiamine (vitamin B1) is synthesized de novo by certain yeast, fungi, plants, protozoans, bacteria and archaea. The pathway of thiamine biosynthesis by archaea is poorly understood, particularly the route of sulfur relay to form the thiazole ring. Archaea harbor structural homologs of both the bacterial (ThiS-ThiF) and eukaryotic (THI4) proteins that mobilize sulfur to thiazole ring precursors by distinct mechanisms.

Results: Based on comparative genome analysis, halophilic archaea are predicted to synthesize the pyrimidine moiety of thiamine by the bacterial pathway, initially suggesting that also a bacterial ThiS-ThiF type mechanism for synthesis of the thiazole ring is used in which the sulfur carrier ThiS is first activated by ThiF-catalyzed adenylation. The only ThiF homolog of Haloferax volcanii (UbaA) was deleted but this had no effect on growth in the absence of thiamine. Usage of the eukaryotic THI4-type sulfur relay was initially considered less likely for thiamine biosynthesis in archaea, since the active-site cysteine residue of yeast THI4p that donates the sulfur to the thiazole ring by a suicide mechanism is replaced by a histidine residue in many archaeal THI4 homologs and these are described as D-ribose-1,5-bisphosphate isomerases. The THI4 homolog of the halophilic archaea, including Hfx. volcanii (HVO_0665, HvThi4) was found to differ from that of methanogens and thermococci by having a cysteine residue (Cys165) corresponding to the conserved active site cysteine of yeast THI4p (Cys205). Deletion of HVO_0665 generated a thiamine auxotroph that was trans-complemented by a wild-type copy of HVO_0665, but not the modified gene encoding an HvThi4 C165A variant.

Conclusions: Based on our results, we conclude that the archaeon Hfx. volcanii uses a yeast THI4-type mechanism for sulfur relay to form the thiazole ring of thiamine. We extend this finding to a relatively large group of archaea, including haloarchaea, ammonium oxidizing archaea, and some methanogen and Pyrococcus species, by observing that these organisms code for THI4 homologs that have a conserved active site cysteine residue which is likely used in thiamine biosynthesis. Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.

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The THI4 homolog (HvThi4) and its C165A variant are synthesized with C-terminal StrepII tags as stable derivatives inHaloferax volcanii. Production of the HvThi4-StrepII and HvThi4 C165A-StrepII proteins in the trans complemented strains (NC1011-pJAM2821 and -pJAM2822, respectively) was detected by anti-StrepII Western blotting (0.065 OD600 units of cells per lane). Equivalent protein loading was assessed by staining blots with Ponceau Red S (not shown) and parallel SDS-PAGE gels with Coomassie Blue R-250 (CB stain, 37–50 kDa range of gel shown).
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Fig5: The THI4 homolog (HvThi4) and its C165A variant are synthesized with C-terminal StrepII tags as stable derivatives inHaloferax volcanii. Production of the HvThi4-StrepII and HvThi4 C165A-StrepII proteins in the trans complemented strains (NC1011-pJAM2821 and -pJAM2822, respectively) was detected by anti-StrepII Western blotting (0.065 OD600 units of cells per lane). Equivalent protein loading was assessed by staining blots with Ponceau Red S (not shown) and parallel SDS-PAGE gels with Coomassie Blue R-250 (CB stain, 37–50 kDa range of gel shown).

Mentions: We next wanted to confirm that the thiamine auxotrophy of the Hfx. volcanii Δthi4 mutant was directly attributed to the thi4 deletion by complementation analysis. We also wanted to analyze the influence of the conserved cysteine residue (Cys165) on HvThi4 activity. To answer these questions, the Δthi4 mutant strain was transformed with plasmids encoding wild-type and C165A variant forms of HvThi4, and the transformed strains were compared to Δthi4 and parent H26 for growth on thiamine minus medium (Figure 4A-B). The plasmid expressed forms of HvThi4 contained a C-terminal StrepII tag to facilitate detection of the protein produced in Hfx. volcanii by anti-StrepII Western blotting. Growth of the Δthi4 mutant strain trans complemented with a wild-type copy of thi4 was similar to the parent H26, revealing that the thiamine auxotrophy is due to the absence of thi4 and not a distal effect of the in-frame deletion of this gene. Addition of the C-terminal StrepII tag had no apparent impact on this trans complementation suggesting HvThi4-StrepII is activated. In contrast to wild type HvThi4, the gene encoding the HvThi4 C165A variant was unable to trans complement the Δthi4 mutation. Western blotting against the C-terminal StrepII tag of HvThi4 (Figure 5) confirmed that the wild-type and C165A variant forms of HvThi4 were produced as proteins at similar levels in the cell suggesting that the differences in complementation are not due to HvThi4 protein levels and instead are based on enzyme activity. Thus, the conserved active site cysteine (Cys165) of HvThi4 is likely required for catalytic activity similarly to THI4p Cys205 of yeast.Figure 5


Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii.

Hwang S, Cordova B, Chavarria N, Elbanna D, McHugh S, Rojas J, Pfeiffer F, Maupin-Furlow JA - BMC Microbiol. (2014)

The THI4 homolog (HvThi4) and its C165A variant are synthesized with C-terminal StrepII tags as stable derivatives inHaloferax volcanii. Production of the HvThi4-StrepII and HvThi4 C165A-StrepII proteins in the trans complemented strains (NC1011-pJAM2821 and -pJAM2822, respectively) was detected by anti-StrepII Western blotting (0.065 OD600 units of cells per lane). Equivalent protein loading was assessed by staining blots with Ponceau Red S (not shown) and parallel SDS-PAGE gels with Coomassie Blue R-250 (CB stain, 37–50 kDa range of gel shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4215014&req=5

Fig5: The THI4 homolog (HvThi4) and its C165A variant are synthesized with C-terminal StrepII tags as stable derivatives inHaloferax volcanii. Production of the HvThi4-StrepII and HvThi4 C165A-StrepII proteins in the trans complemented strains (NC1011-pJAM2821 and -pJAM2822, respectively) was detected by anti-StrepII Western blotting (0.065 OD600 units of cells per lane). Equivalent protein loading was assessed by staining blots with Ponceau Red S (not shown) and parallel SDS-PAGE gels with Coomassie Blue R-250 (CB stain, 37–50 kDa range of gel shown).
Mentions: We next wanted to confirm that the thiamine auxotrophy of the Hfx. volcanii Δthi4 mutant was directly attributed to the thi4 deletion by complementation analysis. We also wanted to analyze the influence of the conserved cysteine residue (Cys165) on HvThi4 activity. To answer these questions, the Δthi4 mutant strain was transformed with plasmids encoding wild-type and C165A variant forms of HvThi4, and the transformed strains were compared to Δthi4 and parent H26 for growth on thiamine minus medium (Figure 4A-B). The plasmid expressed forms of HvThi4 contained a C-terminal StrepII tag to facilitate detection of the protein produced in Hfx. volcanii by anti-StrepII Western blotting. Growth of the Δthi4 mutant strain trans complemented with a wild-type copy of thi4 was similar to the parent H26, revealing that the thiamine auxotrophy is due to the absence of thi4 and not a distal effect of the in-frame deletion of this gene. Addition of the C-terminal StrepII tag had no apparent impact on this trans complementation suggesting HvThi4-StrepII is activated. In contrast to wild type HvThi4, the gene encoding the HvThi4 C165A variant was unable to trans complement the Δthi4 mutation. Western blotting against the C-terminal StrepII tag of HvThi4 (Figure 5) confirmed that the wild-type and C165A variant forms of HvThi4 were produced as proteins at similar levels in the cell suggesting that the differences in complementation are not due to HvThi4 protein levels and instead are based on enzyme activity. Thus, the conserved active site cysteine (Cys165) of HvThi4 is likely required for catalytic activity similarly to THI4p Cys205 of yeast.Figure 5

Bottom Line: The THI4 homolog of the halophilic archaea, including Hfx. volcanii (HVO_0665, HvThi4) was found to differ from that of methanogens and thermococci by having a cysteine residue (Cys165) corresponding to the conserved active site cysteine of yeast THI4p (Cys205).Based on our results, we conclude that the archaeon Hfx. volcanii uses a yeast THI4-type mechanism for sulfur relay to form the thiazole ring of thiamine.Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, 32611-0700, USA. sungminhwang@ufl.edu.

ABSTRACT

Background: Thiamine (vitamin B1) is synthesized de novo by certain yeast, fungi, plants, protozoans, bacteria and archaea. The pathway of thiamine biosynthesis by archaea is poorly understood, particularly the route of sulfur relay to form the thiazole ring. Archaea harbor structural homologs of both the bacterial (ThiS-ThiF) and eukaryotic (THI4) proteins that mobilize sulfur to thiazole ring precursors by distinct mechanisms.

Results: Based on comparative genome analysis, halophilic archaea are predicted to synthesize the pyrimidine moiety of thiamine by the bacterial pathway, initially suggesting that also a bacterial ThiS-ThiF type mechanism for synthesis of the thiazole ring is used in which the sulfur carrier ThiS is first activated by ThiF-catalyzed adenylation. The only ThiF homolog of Haloferax volcanii (UbaA) was deleted but this had no effect on growth in the absence of thiamine. Usage of the eukaryotic THI4-type sulfur relay was initially considered less likely for thiamine biosynthesis in archaea, since the active-site cysteine residue of yeast THI4p that donates the sulfur to the thiazole ring by a suicide mechanism is replaced by a histidine residue in many archaeal THI4 homologs and these are described as D-ribose-1,5-bisphosphate isomerases. The THI4 homolog of the halophilic archaea, including Hfx. volcanii (HVO_0665, HvThi4) was found to differ from that of methanogens and thermococci by having a cysteine residue (Cys165) corresponding to the conserved active site cysteine of yeast THI4p (Cys205). Deletion of HVO_0665 generated a thiamine auxotroph that was trans-complemented by a wild-type copy of HVO_0665, but not the modified gene encoding an HvThi4 C165A variant.

Conclusions: Based on our results, we conclude that the archaeon Hfx. volcanii uses a yeast THI4-type mechanism for sulfur relay to form the thiazole ring of thiamine. We extend this finding to a relatively large group of archaea, including haloarchaea, ammonium oxidizing archaea, and some methanogen and Pyrococcus species, by observing that these organisms code for THI4 homologs that have a conserved active site cysteine residue which is likely used in thiamine biosynthesis. Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.

Show MeSH
Related in: MedlinePlus